Supplementary Material for: Influences of Various Peptide Linkers on the Thermotoga maritima MSB8 Nitrilase Displayed on the Spore Surface of Bacillus subtilis
Chen H.
Chen Z.
Wu B.
Ullah J.
Zhang T.
Jia J.
Wang H.
Tan T.
10.6084/m9.figshare.4564921.v1
https://karger.figshare.com/articles/dataset/Supplementary_Material_for_Influences_of_Various_Peptide_Linkers_on_the_Thermotoga_maritima_MSB8_Nitrilase_Displayed_on_the_Spore_Surface_of_Bacillus_subtilis/4564921
<p>In the present study, fusion genes composed of <i>Thermotoga maritima</i> MSB8 nitrilase and <i>Bacillus subtilis</i> 168 outer coat protein CotG were constructed with various peptide linkers and displayed on <i>B. subtilis</i> DB 403 spores. The successful display of CotG-nit fusion proteins on the spore surface of <i>B. subtilis</i>
was verified by Western blot analysis and activity measurement. It was
demonstrated that the fusion with linker GGGGSEAAAKGGGGS presented the
highest thermal and pH stability, which is 2.67- and 1.9-fold of the
fusion without linker. In addition, fusion with flexible linker (GGGGS)<sub>3</sub> demonstrated better thermal and pH stability than fusions with linkers GGGGS and (GGGGS)<sub>2</sub>. Fusion with rigid linker (EAAAK) demonstrated better thermal stability than fusions with linkers (EAAAK)<sub>2</sub> and (EAAAK)<sub>3</sub>. Fusions with linker (EAAAK)<sub>2</sub> demonstrated better pH stability than fusions with linkers (EAAAK) and (EAAAK)<sub>3</sub>. In the presence of 1 mM
dithiothreitol, 1% (v/v) sodium dodecyl sulfate, and 20% (v/v) ethanol,
the optimal linkers of the fusions were MGSSSN, GGGGSEAAAKGGGGS, and
(GGGGS)<sub>3</sub>, respectively. In summary, our results showed that
optimizing the peptide linkers with different type, length, and amino
acid composition of the fusion proteins would be an efficient way to
maintain the stability of fusion proteins and thus improve the nitrilase
display efficiency, which could provide an effective method for
rational design peptide linkers of displayed nitrilase on <i>B. subtilis</i>.</p>
2017-01-19 13:19:42
Fusion protein
Nitrilase
Linker peptide
Thermal and pH stability